Use of nitrooxy organic molecules in feed for reducing methane emission in ruminants, and/or to improve ruminant performance

ABSTRACT

The present invention relates to a method for reducing the production of methane emanating from the digestive activities of a ruminant and/or for improving ruminant animal performance by using, as active compound at least one organic molecule substituted at any position with at least one nitrooxy group, or a salt thereof, which is administrated to the animal together with the feed. The invention also relates to the use of these compounds in feed and feed additives such as premix, concentrates and total mixed ration (TMR) or in the form of a bolus.

This application is a divisional of commonly owned U.S. application Ser.No. 13/996,026, filed Sep. 23, 2013 (now U.S. Pat. No. 9,266,814), whichis the national phase application under 35 USC §371 ofPCT/EP2011/072707, filed Dec. 20, 2011 which designated the US andclaims benefit of European Application No. 10195857.7, filed Dec. 20,2010 and European Application No. 11178994.7, filed Aug. 26, 2011, theentire contents of each of which are hereby incorporated by reference.

The present invention relates to the use of at least one organicmolecule substituted at any position with at least one nitrooxy groupfor reducing the production of methane emanating from the digestiveactivities of ruminants, and/or to improve the ruminant performance.

The present invention also relates to animal feed or animal feedcompositions and feed additives comprising the above mentionedmolecules. The term feed or feed composition means any compound,preparation, mixture, or composition suitable for, or intended forintake by an animal.

In the present context, a ruminant is a mammal of the order Artiodactylathat digests plant-based food by initially softening it within theanimal's first stomach, known as the rumen, then regurgitating thesemi-digested mass, now known as cud, and chewing it again. The processof again chewing the cud to further break down plant matter andstimulate digestion is called “ruminating”.

Rumen fermentation brings some disadvantages. Methane is produced as anatural consequence of the anaerobic fermentation, which represents anenergy loss to the host animal. Carbohydrate makes up 70-80% of the drymatter in a typical dairy cattle ration and in spite of this theabsorption of carbohydrates from the gastro-intestinal tract is normallyvery limited. The reason for this is the extensive fermentation ofcarbohydrates in the rumen resulting in production of acetate,propionate and butyrate as the main products. These products are part ofthe so called volatile fatty acids, (VFAs).

Besides the energy loss, methane is also a greenhouse gas, which is manytimes more potent than CO₂. Its concentration in the atmosphere hasdoubled over the last century and continues to increase alarmingly.Ruminants are the major contributors to the biogenic methane formation,and it has been estimated that the prevention of methane formation fromruminants would almost stabilize atmospheric methane concentrations.

Furthermore, the assessment of the Kyoto protocol followed by theCopenhagen climate summit in 2009 places increased priority indecreasing methane emissions as part of a multi-gas strategy. The mosteffective additives currently used for reducing the formation of methanecontain antibiotics which diminish the proliferation of microorganismsproviding hydrogen (H₂) to the methanogens (Sauer et al. 1998. AmericanSociety of Animal Science; 76: 906-914). However, the effect ofantibiotics on the formation of methane has some disadvantages becauseof rapid adaptation of the microflora and/or resistance developmentleading to a complete loss of the intended effect within a short periodof time (2 to 3 weeks), and because the use of antibiotics is banned inEurope for non therapeutic use.

Non antibiotic products (bile acid derivatives) leading to reduction ofmethane emission, when tested using an in vitro rumen simulation model,have recently been published (WO 2010072584). However, the amountrequired to produce a moderate reduction of methane emission are notcompatible with the ruminant feed industry cost constraints.

Furthermore, a number of natural plant extracts (Garlic: WO 2009150264,yucca, cinnamon, rhubarb . . . ) have been described in the scientificliterature as potent solutions to reduce methane emission in ruminantsbased on in vitro experiments. However, none of these solutions made itto a commercial product because of side effects (residues in milk),because of lack efficacy, when tested in vivo, or because of the verylarge amount of additive which needs to be supplied to the animal togenerate a significant methane reduction.

Under these circumstances there is still a need to develop newsubstances which reduce the formation of methane and which are in linewith reliable and generally accepted practice and not of a medicinalnature. In addition to reducing methane emission, such substances mayalso contribute to improve ruminant performance by improving the feedconversion ratio, reducing feed intake, improving weight gain, and/orimproving carcass, or milk yield.

The present inventors now surprisingly found that the compoundsspecified herein after, have a great potential for use in animal feed inorder to essentially reduce the formation of methane without affectingmicrobial fermentation in a way that would be detrimental to the hostanimal. Moreover, the compounds of the present invention also have agreat benefit regarding overall animal performance as measured by feedconversion ratio, feed intake, weight gain, carcass yield, or milkyield. Said compounds are also more stable than those described in theprior art, safer for the animal and human, lead to persistent methanereduction effect, they do not affect palatability, they can be producedat industrial scale at a cost compatible with the animal nutritionindustry, and above all, they do not provoke accumulation of anymetabolite in the milk or meat of the supplemented animal, and they areactive at very low concentration in the rumen.

In particular, the present inventors have observed that the feeding toruminants of at least one organic molecule substituted at any positionwith at least one nitrooxy group is very effective for reducing theproduction of methane emanating from the digestive activities ofruminants without negatively affecting total VFA production, and/or forimproving the ruminant performance. Moreover, the present inventors haveshown that when the nitrooxy group is replaced by other chemical groupsof similar physicochemical properties, the technical effect on methaneproduction is lost demonstrating that the Nitrooxy group is key for theeffect on methane reduction of the present invention.

It is known from the international patent application Nr.:PCT/EP2010/069338 that nitrooxy-carboxylic acid derivatives are potentinhibitors of rumen methanogenesis in vitro, and also in vivo.Therefore, these molecules are specifically disclaimed from the presentinvention.

Therefore, the present invention provides the use of at least oneorganic molecule substituted at any position with at least one nitrooxygroup, or a salt thereof as defined by formula (I) as an active compoundin animal feeding for reducing the formation of methane emanating fromthe digestive activities of ruminants and/or for improving ruminantperformance.

The invention further provides a method for reducing the production ofmethane emanating from the digestive activities of ruminants and/or forimproving ruminant animal performance, comprising orally administering asufficient amount of at least one organic molecule substituted at anyposition with at least one nitrooxy group, or a salt thereof as definedby formula (I) to the animal. It is to be understood by oraladministration a simple feeding, or manual administration of a bolus.

In all embodiments of the present invention, organic moleculessubstituted at any position with at least one nitrooxy group, or saltsthereof are defined by the following compound of formula (I)

wherein Y is an organic molecule of the following composition:C_(a)H_(b)O_(d)N_(e)S_(g),wherein

-   a is comprised between 1 and 25, preferably between 1 and 10-   b is comprised between 2 and 51, preferably between 2 and 21-   d is comprised between 0 and 8, preferably between 0 and 6-   e is comprised between 0 and 5, preferably between 0 and 3-   g is comprised between 0 and 3, preferably between 0 and 1,    wherein nitrooxy alkanoic acid, and/or derivatives thereof as    defined by the formula (II) are excluded,

wherein

-   u is comprised between 0 and 23 and, wherein if u≠0, the carbon    chain is a linear, a cyclic, or branched linear or cyclic aliphatic    carbon chain which may be mono- or polyunsaturated and in any    isomeric form,-   Z is independently O, NH, or N—R3, wherein if R1≠H, Z—R1 represents    an ester or a secondary amide derivative,-   R1 is independently, hydrogen or a saturated straight, cyclic or    branched chain of an alkyl or alkenyl group containing 1 to 10    carbon atoms,-   R2 is independently, hydrogen or a saturated straight or branched    chain of an alkyl or alkenyl group containing 1 to 23 carbon atoms,    and-   R3 is independently, hydrogen or a saturated straight, cyclic or    branched chain of an alkyl or alkenyl group containing 1 to 10    carbon atoms.

In another embodiment, preferred compounds of formula (I) according tothe present invention are compounds, wherein a is comprised between 1and 10, preferably, a is comprised between 3 and 8.

In another embodiment, preferred compounds of formula (I) according tothe present invention are compounds of formula (III),

wherein

-   n is comprised between 0 and 12, preferably comprised between 0 and    6 and, wherein, if n≠0, the carbon chain is a linear, a cyclic, or    branched aliphatic carbon chain which may be non substituted or    substituted with up to 3 hydroxyl-, alkoxy-, amino-, alkylamino-,    dialkylamino- or nitrooxy groups, or an alkenyl, or an alkynyl    carbon chain mono- or polyunsaturated and in any isomeric form,-   R4 is independently, hydrogen or a saturated straight, cyclic or    branched chain of an alkyl or alkenyl group containing 1 to 12,    preferably 1 to 6 carbon atoms,-   X is hydrogen, R5, R5≡N, —OR5, —OCOR5, —NR5R6, —ONO2, —COOR5,    —CONR5R6, —NHSO2R5, or —SO2NHR5,-   R5 and R6 are independently, hydrogen, C1-C12 straight, branched or    cyclic alkyl chain, non substituted or substituted with up to 3    hydroxyl-, alkoxy-, amino-, alkylamino-, dialkylamino- or nitrooxy    groups, alkenyl, or alkynyl carbon chain which may be mono or    polyunsaturated, and in any isomeric form.

For all embodiments of the present invention, it is to be understoodthat compounds of formula (I) and compounds of formula (III) can be inany isomeric form.

It is to be understood in the above definition of compounds of formula(III) that when n>2, the carbon chain can be linear or branched at anyposition along the carbon chain. In addition, the carbon chain can bebranched by multiple branches at different positions along the carbonchain. Moreover, when n>3, the aliphatic carbon chain may form a cyclicmoiety. This cyclic moiety can carry the nitrooxy moiety at any position(2, 3, 4), and it can also be branched at multiple positions by anyaliphatic groups. The branched aliphatic groups are preferably, methyl,ethyl or propyl. Moreover, the carbon chain may be further substitutedwith up to 3 hydroxyl-, alkoxy-, amino-, alkylamino-, dialkylamino- ornitrooxy groups.

In the above definition of derivatives of the formula (III) a preferredalkyl group is methyl, ethyl, propyl, isopropyl, butyl, sec. butyl,isobutyl, pentyl, neopentyl, hexyl, cyclohexyl, and 2-ethyl-hexyl andoctyl. Furthermore any alkyl or alkenyl group containing three or morecarbon atoms can be straight chain, branched, or cyclic. In addition forthe straight chain or branched C₂-C₁₀-alkenylene group, this isunderstood to encompass alkenylene groups with one or (from C₄) moredouble bonds; examples of such alkenylene groups are those of theformulae —CH═CH—, —CH═CH—CH₂—, —CH═CH—(CH₂)₃— and —(CH═CH)₂—.

In another embodiment, more preferred compounds of formula (I) accordingto the present invention are selected from the list of compounds, andsalts thereof comprising: 3-Nitrooxypropanol,racemate-4-Phenylbutane-1,2-diyl dinitrate,2-(Hydroxymethyl)-2-(nitrooxymethyl)-1,3-propanediol,N-Ethyl-3-nitrooxy-propionic sulfonyl amide, 5-Nitrooxy-pentanenitrile,5-Nitrooxy-pentane, 3-Nitrooxy-propyl propionate,1,3-bis-Nitrooxypropane, 1,4-bis-Nitrooxybutane,1,5-bis-Nitrooxypentane, 3-Nitrooxy-propyl benzoate, 3-Nitrooxy-propylhexanoate, 3-Nitrooxy-propyl 5-nitrooxy-hexanoate, Benzylnitrate,isosorbid-dinitrate, and N-[2-(Nitrooxy)ethyl]-3-pyridinecarboxamide,2-Nitro-5-n itrooxymethyl-furan, and Bis-(2-nitrooxyethyl) ether aslisted in Table 1:

TABLE 1 Preferred compounds of formula (I) according to the presentinvention Comp. Identifier Molecular structure Chemical name 1

3-Nitrooxypropanol 2

rac-4-Phenylbutane-1,2- diyl dinitrate 3

2-(Hydroxymethyl)-2- (nitrooxymethyl)-1,3- propanediol 4

N-Ethyl-3-nitrooxy- propionic sulfonyl amide 5

5-Nitrooxy-pentanenitrile 6

5-Nitrooxy-pentane 7

3-Nitrooxy-propyl propionate 8

1,3-bis-Nitrooxypropane 9

1,4-bis-Nitrooxybutane 10

1,5-bis-Nitrooxypentane 11

3-Nitrooxy-propyl benzoate 12

3-Nitrooxy-propyl hexanoate 13

3-Nitrooxy-propyl 5- nitrooxy-hexanoate 14

Benzylnitrate 15

isosorbid-dinitrate 16

N-[2-(Nitrooxy)ethyl]-3- pyridinecarboxamide 17

2-Nitro-5-nitrooxymethyl- furan 18

Bis-(2-nitrooxyethyl) ether

In another embodiment, even more preferred compounds of formula (III)based on the strength of their effect in reducing methane are selectedfrom the list of compounds, and salts thereof comprising:3-Nitrooxypropanol, 5-Nitrooxy-pentanenitrile, 5-Nitrooxy-pentane,3-Nitrooxy-propyl propionate, 1,3-bis-Nitrooxypropane,1,4-bis-Nitrooxybutane, 1,5-bis-Nitrooxypentane, 3-Nitrooxy-propylbenzoate, 3-Nitrooxy-propyl hexanoate, 3-Nitrooxy-propyl5-nitrooxy-hexanoate, isosorbid-dinitrate, andN-[2-(Nitrooxy)ethyl]-3-pyridinecarboxamide, and Bis-(2-nitrooxyethyl)ether as listed in Table 2:

TABLE 2 Most preferred compounds of formula (I) according to the presentinvention Comp. Identifier Molecular structure Chemical name 1

3-Nitrooxypropanol 5

5-Nitrooxy-pentanenitrile 6

5-Nitrooxy-pentane 7

3-Nitrooxy-propyl propionate 8

1,3-bis-Nitrooxypropane 9

1,4-bis-Nitrooxybutane 10

1,5-bis-Nitrooxypentane 11

3-Nitrooxy-propyl benzoate 12

3-Nitrooxy-propyl hexanoate 13

3-Nitr-oxy-propyl 5- nitrooxy-hexanoate 15

Isosorbid-dinitrate 16

N-[2-(Nitrooxy)ethyl]-3- pyridinecarboxamide 18

Bis-(2-nitrooxyethyl) ether

In another embodiment, most preferred compound of formula (I) based onthe strength of their effect in reducing methane and on the productionprocess is a mixture of 3-nitrooxy propanol and 1,3-bis-nitrooxypropane.Preferably the ratio 3-nitrooxy propanol/1,3-bis-nitrooxypropane iscomprised between 1/10 and 1000/1, more preferably, between 1/5 and100/1, most preferably, between 1/1 and 10/1.

The compounds of the present invention also comprise salts of thenitrooxy organic molecule. Preferred cations for salt preparation may beselected from the group consisting of sodium (Na+), potassium (K+),lithium (Li+), magnesium (Mg2+), calcium (Ca2+), barium (Ba2+),strontium (Sr2+), and ammonium (NH4+). Salts may also be prepared froman alkali metal or an alkaline earth metal.

The compounds of the present invention can be manufactured in principleaccording to synthetic methods known per se for nitrooxy organicmolecules, and/or based on methods as described in the examples.

In all these cases appropriate methods to purify the product (compoundsof formula (I)) can be chosen by those skilled in the art, i.e. bycolumn chromatography, or the compound of formula (I), can be isolatedand purified by methods known per se, e.g. by adding a solvent such asdiethyl-ether or ethyl acetate to induce the separation of the crudeproduct from the mixture after reaction, and drying over Na₂SO₄ of thecollected crude product.

Methane emission by ruminants can easily be measured in individualanimals in metabolic chambers by methods known in the art (Grainger etal., 2007 J. Dairy Science; 90: 2755-2766). Moreover, it can also beassessed at barn level by an emerging technology using laser beam(McGinn et al., 2009, Journal of Environmental Quality; 38: 1796-1802).Alternatively, methane produced by a dairy ruminant can also be assessedby measurement of VFA profiles in milk according to WO 2009/156453.

Ruminant performance can be assessed by methods well known in the art,and is usually characterized by feed conversion ratio, feed intake,weight gain, carcass yield, or milk yield.

The present invention also relates to the use of at least one organicmolecule substituted at any position with at least one nitrooxy group,or a salt thereof as defined by formula (I) in combination with at leastone additional active substance which shows similar effects with regardto methane formation in the rumen and which is selected from the groupconsisting of diallyl disulfide, garlic oil, allyl isothiocyanate,deoxycholic acid, chenodeoxycholic acid and derivatives thereof.

Further components that could be given together with the compoundaccording to the present invention are for example yeasts, essentialoils, and ionophores like Monensin, Rumensin.

It is at present contemplated that diallyl disulfide, garlic oil, allylisothiocyanate deoxycholic acid, chenodeoxycholic acid and derivativesthereof are independently administered in dosage ranges of for example0.01-500 mg active substance per kg feed (ppm). These compounds areeither commercially available or can easily be prepared by a skilledperson using processes and methods well-known in the prior art.

Ruminating mammals according to the present invention include cattle,goats, sheep, giraffes, American Bison, European bison, yaks, waterbuffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn,and nilgai.

For all embodiments of the present invention, domestic cattle, sheep andgoat are the more preferred species. For the present purposes mostpreferred species are domestic cattle. The term includes all races ofdomestic cattle, and all production kinds of cattle, in particular dairycows and beef cattle.

The present invention also relates to the use of at least one organicmolecule substituted at any position with at least one nitrooxy group,or a salt thereof as defined by formula (I), wherein the methaneproduction in ruminants calculated in liters per kilogram of dry matterintake is reduced by at least 10% when measured in metabolic chambers.Preferably, methane reduction is at least 15%, more preferably, at least20%, even more preferably, at least 25%, most preferably, at least 30%.Alternative methane emission measurements may also be used like using alaser beam or for dairy ruminants, correlating methane production to theVFA profile in milk.

The present invention also relates to the use of at least one organicmolecule substituted at any position with at least one nitrooxy group,or a salt thereof as defined by formula (I), wherein the ruminant feedconversion ratio is reduced by at least 1% when measured in conventionalperformance trial. Preferably, the feed conversion ratio is reduced byat least 2%, more preferably, by at least 2.5%, even more preferably, byat least 3%, most preferably, by at least 3.5%.

The present invention also relates to the use of at least one organicmolecule substituted at any position with at least one nitrooxy group,or a salt thereof as defined by formula (I), wherein the amount of theat least one active compound as defined in formula (I) administered tothe ruminant animal is from 1 mg to 10 g per Kg of feed, preferably from10 mg to 1 g per Kg of feed, more preferably, from 50 mg to 500 mg perKg of feed. For the use in animal feed, however, organic moleculessubstituted at any position with at least one nitrooxy group, or theirsalts thereof as defined by formula (I) need not be that pure; it maye.g. include other compounds and derivatives.

As indicated above, the compounds of the present invention are useful ascompounds for feed additives and animal feed compositions for ruminants,and accordingly are useful as the active ingredients in such feed toreduce methane formation in the digestive tract of the animal, and/or toimprove ruminant performance.

For the realisation of their use as such ingredients for the feed ofruminants the compounds may be incorporated in the feed by methods knownper se in the art of feed formulation and processing.

Further aspects of the present invention are therefore formulations,i.e. feed additives and animal feed compositions containing compounds asherein above defined.

The present invention therefore also relates to a feed composition or afeed additive comprising at least one compound of formula (I) or a saltthereof. Preferably, the feed composition or feed additive is a ruminantbase mix. In a preferred embodiment, the composition is a mineralpremix, a vitamin premix including vitamins and minerals or a bolus.

The normal daily dosage of a compound according to the inventionprovided to an animal by feed intake depends upon the kind of animal andits condition. Normally this dosage should be in the range of from about1 mg to about 10 g, preferably from about 10 mg to about 1 g, morepreferably, 50 mg to 500 mg compound per kg of feed.

The at least one organic molecule substituted at any position with atleast one nitrooxy group, or a salt thereof as defined by formula (I)may be used in combination with conventional ingredients present in ananimal feed composition (diet) such as calcium carbonates, electrolytessuch as ammonium chloride, proteins such as soya bean meal, wheat,starch, sunflower meal, corn, meat and bone meal, amino acids, animalfat, vitamins and trace minerals.

Particular examples of compositions of the invention are the following:

-   -   An animal feed additive comprising (a) at least one compound        selected from table 1 and (b) at least one fat-soluble        vitamin, (c) at least one water-soluble vitamin, (d) at least        one trace mineral, and/or (e) at least one macro mineral;    -   An animal feed composition comprising at least one compound        selected from table 1 and a crude protein content of 50 to 800        g/kg feed.

Therefore, in a preferred embodiment, the present invention relates to aruminant feed composition or feed additive

The so-called premixes are examples of animal feed additives of theinvention. A premix designates a preferably uniform mixture of one ormore micro-ingredients with diluents and/or carrier. Premixes are usedto facilitate uniform dispersion of micro-ingredients in a larger mix.

Apart from the active ingredients of the invention, the premix of theinvention contains at least one fat-soluble vitamin, and/or at least onewater soluble vitamin, and/or at least one trace mineral, and/or atleast one macro mineral. In other words, the premix of the inventioncomprises the at least one compound according to the invention togetherwith at least one additional component selected from the groupconsisting of fat-soluble vitamins, water-soluble vitamins, traceminerals, and macro minerals.

Macro minerals may be separately added to the feed. Therefore, in aparticular embodiment, the premix comprises the active ingredients ofthe invention together with at least one additional component selectedfrom the group consisting of fat-soluble vitamins, water-solublevitamins, and trace-minerals.

The following are non-exclusive lists of examples of these components:

-   -   Examples of fat-soluble vitamins are vitamin A, vitamin D3,        vitamin E, and vitamin K, e.g. vitamin K3.    -   Examples of water-soluble vitamins are vitamin B12, biotin and        choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid        and panthothenate, e.g. Ca—D-panthothenate.    -   Examples of trace minerals are manganese, zinc, iron, copper,        iodine, selenium, and cobalt.    -   Examples of macro minerals are calcium, phosphorus and sodium.

As regards feed compositions for ruminants such as cows, as well asingredients thereof, the ruminant diet is usually composed of an easilydegradable fraction (named concentrate) and a fiber-rich less readilydegradable fraction (named hay, forage, or roughage).

Hay is made of dried grass, legume or whole cereals. Grasses includeamong others timothy, ryegrasses, fescues. Legumes include among othersclover, lucerne or alfalfa, peas, beans and vetches. Whole cerealsinclude among others barley, maize (corn), oat, sorghum. Other foragecrops include sugarcane, kales, rapes, and cabbages. Also root cropssuch as turnips, swedes, mangles, fodder beet, and sugar beet (includingsugar beet pulp and beet molasses) are used to feed ruminants. Stillfurther crops are tubers such as potatoes, cassava and sweet potato.Silage is an ensiled version of the fiber-rich fraction (e.g. fromgrasses, legumes or whole cereals) whereby material with a high watercontent is treated with a controlled anaerobic fermentation process(naturally-fermented or additive treated).

Concentrate is largely made up of cereals (such as barley includingbrewers grain and distillers grain, maize, wheat, sorghum), but alsooften contain protein-rich feed ingredients such as soybean, rapeseed,palm kernel, cotton seed and sunflower.

Cows may also be fed total mixed rations (TMR), where all the dietarycomponents, e.g. forage, silage and concentrate, are mixed beforeserving.

As mentioned above a premix is an example of a feed additive which maycomprise the active compounds according to the invention. It isunderstood that the compounds may be administered to the animal indifferent other forms. For example the compounds can also be included ina bolus that would be placed in the rumen and that would release adefined amount of the active compounds continuously in well defineddosages over a specific period of time.

The present invention further relates to a method for reducing theproduction of methane emanating from the digestive activities ofruminants and/or for improving ruminant animal performance, comprisingorally administering a sufficient amount of at least one organicmolecule substituted at any position with at least one nitrooxy group,or a salt thereof as defined by formula (I) with the preferredembodiments described above.

Moreover, the invention further relates to a method as described above,wherein the compound of formula (I) is administered to the animal incombination with at least one additional active substance selected fromthe group consisting of diallyl disulfide, garlic oil, allylisothiocyanate, deoxycholic acid, chenodeoxycholic acid and derivativesthereof.

The invention also relates to a method as described above, wherein theruminant animal is selected from the group consisting of: cattle, goats,sheep, giraffes, American Bison, European bison, yaks, water buffalo,deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, andnilgai, and more preferably from the group consisting of: cattle, goatsand sheep.

The invention also relates to a method as described above, wherein theamount of the at least one active compound as defined in formula (I)administered to the ruminant animal is from about 1 mg to about 10 g perkg feed, preferably from about 10 mg to about 1 g, more preferably from50 mg to 500 mg compound per kg of feed.

The invention also relates to a method as described above, wherein themethane production in ruminants calculated in liters per kilogram of drymatter intake is reduced by at least 10% when measured in metabolicchambers. Preferably, methane reduction is at least 15%, morepreferably, at least 20%, even more preferably, at least 25%, mostpreferably, at least 30%. Alternative methane emission measurements mayalso be used like using a laser beam or for dairy ruminants, correlatingmethane production to the VFA profile in milk.

The invention also relates to a method as described above, wherein theruminant feed conversion ratio is reduced by at least 1% when measuredin conventional performance trial. Preferably, the feed conversion ratiois reduced by at least 2%, more preferably, by at least 2.5%, even morepreferably, by at least 3%, most preferably, by at least 3.5%.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Example 1 In Vitro Test for Methane Production

A modified version of the “Hohenheim Forage value Test (HFT)” was usedfor testing the effect of specific compounds on the rumen functionsmimicked by this in-vitro system.

Principle:

Feed is given into a syringe with a composition of rumen liquor and anappropriate mixture of buffers. The solution is incubated at 39° C.After 8 hours the quantity (and composition) of methane produced ismeasured and put into a formula for conversion.

Reagents:

Mass Element Solution:

-   -   6.2 g potassium dihydrogen phosphate (KH₂PO₄)    -   0.6 g magnesium sulfate heptahydrate (MgSO₄*7H₂O)    -   9 ml concentrated phosphoric acid (1 mol/l)    -   dissolved in distilled water to 1 l (pH about 1.6)        Buffer Solution:    -   35.0 g sodium hydrogen carbonate (NaHCO₃)    -   4.0 g ammonium hydrogen carbonate ((NH₄)HCO₃)    -   dissolved in distilled water to 1 l        Trace Element Solution:    -   13.2 g calcium chloride dihydrate (CaCl₂*2H₂O)    -   10.0 g manganese(II) chloride tetrahydrate (MnCl₂*4H₂O)    -   1.0 g cobalt(II) chloride hexahydrate (CoCl₂*6H₂O)    -   8.0 g iron(III) chloride (FeCl₃*6H₂O)    -   dissolved in distilled water to 100 ml        Sodium Salt Solution:    -   100 mg sodium salt    -   dissolved in distilled water to 100 ml        Reduction Solution:    -   first 3 ml sodium hydroxide (c=1 mol/l), then 427.5 mg sodium        sulfide hydrate (Na₂S*H₂O) are added to 71.25 ml H₂O    -   solution must be prepared shortly before it is added to the        medium solution        Procedure:        Sample Weighing:

The feed stuff is sieved to 1 mm—usually TMR (44% concentrate, 6% hay,37% maize silage and 13% grass silage)—and weighed exactly into 64syringes. 4 of these syringes are the substrate controls, which displaythe gas production without the effect of the tested compounds. 4 othersyringes are positive control, in which bromoethane sulfonate has beenadded to 0.1 mM. When needed, 4 syringes contain a carrier control (ifthe test compounds need a carrier). The remaining syringes contain thetest substances, by groups of 4 syringes.

Preparation of the Medium Solution:

The components are mixed in a Woulff bottle in following order:

-   -   711 ml water    -   0.18 ml trace element solution    -   355.5 ml buffer solution    -   355.5 ml mass element solution

The completed solution is warmed up to 39° C. followed by the additionof 1.83 ml sodium salt solution and the addition of reduction solutionat 36° C. The rumen liquor is added, when the indicator turnscolourless.

Extraction of the Rumen Liquor:

750 ml of rumen liquor are added to approximately 1,400 ml of mediumsolution under continued agitation and CO₂-gassing.

Filling the Syringes, Incubation and Determining Gas Volumes and VFAValues:

The diluted rumen fluid (24 ml) is added to the glass syringe. Thesyringes are then incubated for 8 hours at 39° C. under gentleagitation. After 8 hours, the volume of gas produced is measured, andthe percentage of methane in the gas phase is determined by gaschromatography.

Results

The food fermented was artificial TMR (44% concentrate, 6% hay, 37%maize silage and 13% grass silage). The compounds produced as describedin examples 2 to 14 were added to the fermentation syringes to aconcentration of 2 to 0.005 of dry matter (DM). The results arepresented in the following table.

TABLE 3 Methane reduction effect resulting from the average of twoexperiments with some compounds according to the present invention (aninteger in the column effect on methanogenesis change (%) means areduction in methane produced when compared to control; no value meansthat the concentration was not tested) effect on methanogenesis (%) 2%1% 0.5% 0.25% 0.1% 0.05% 0.01% 0.005% Structure DM DM DM DM DM DM DM DM

100 100 100 100 79 20

10 4

85 6

99 99 24 10

99 95 12 7

100 100 33 4

100 100 21 6

99 100 98 29

100 100 92 16

100 100 45 6

99 99 11

98 99 42

100 100 37 3

100

100

100 99 64 3

Example 2 Comparative Example: In Vitro Test for Methane Production

The same in vitro assay as described in example 1 has been performedwith a series of molecules, wherein the nitrooxy group has been replacedby different organic groups. Moreover, the inorganic salt Na NO3 hasalso been tested. See results in Table 4. This data demonstrates that asignificant methane reduction activity is only observed when theNitrooxy group is present in the series.

TABLE 4 Methane reduction effect resulting from the average of twoexperiments with 3-nitrooxypropanol according to the present inventionincomparison with similar compounds in which the nitrooxy group has beenreplaced. (An integer in the column effect on methanogenesis change (%)means a reduction in methane produced when compared to control; no valuemeans that the concentration was not tested) effect on methanogenesis(%) 0.5% 0.1% 0.05% 0.01% Structure 2% DM DM DM DM DM

100 100 100 79

2

8

6

2 Na NO3 23 2

Example 3 Synthesis of 3-Nitrooxypropanol

50.1 mmol 3-Bromopropanol dissolved in 100 ml acetonitrile and 125.25mmol silver nitrite were added into a flask protected from light. Thissuspension was stirred for 21 hours at 70° C. After cooling to roomtemperature the suspension was filtrated and concentrated in vacuo. Theresidue was dissolved in Water and extracted two times with TMBE. Theorganic phases were washed with water and brine, combined, dried overNa₂SO₄ and the solvent was removed in vacuo leaving 5.63 g.

The crude product was purified by flash chromatography on silica gelusing heptane/ethyl acetate 2:1; Yield: 4.82 g (38.8 mmol, 77.4%).

Example 4 Synthesis of2-(Hydroxymethyl)-2-(nitrooxymethyl)-1,3-propanediol

5 mmol 2-(Bromomethyl)-2-(hydroxymethyl)-1,3-propanediol dissolved in 20ml acetonitrile and 15 mmol silver nitrite were added into a flaskprotected from light. This suspension was stirred for 24 hours at 70° C.After cooling to room temperature the suspension was filtrated and thesolvent was removed in vacuo leaving 3.05 g.

The crude product was purified by flash chromatography on silica gelusing dichloromethane/methanol 50:1; Yield: 0.36 g (1.99 mmol, 40.2%).

Example 5 Synthesis of rac-4-Phenylbutane-1,2-diyldinitrate

7.5 mmol 4-Phenyl-1-buten dissolved in 40 ml acetonitrile, 20.3 mmolsilver nitrite and 7.5 mmol lode were added into a flask protected fromlight. This suspension was stirred for 30 minutes at 25° C. and then for16 hours at 79° C. After cooling to room temperature the suspension wasfiltrated and washed with Ethyl acetate. The filtrate was extractedthree times with water and washed brine, dried over Na₂SO₄ and thesolvent was removed in vacuo leaving 1.92 g.

The crude product was purified by flash chromatography on silica gelusing Hexane/Ethyl acetate 10:1; Yield: 0.52 g (2.03 mmol, 27%).

Example 6 Synthesis of N-Ethyl-3-nitrooxy-propionic sulfonyl amide

In a flask 17 mmol 3-chloropropionic sulfonyl chloride were dissolved in5 ml Tetrahydrofurane. 33.3 mmol Ethylamine were added over a period of45 minutes. After that, the solvent was removed in vacuo. The residuewas dissolved in water, extracted three times with ethyl acetate. Thecombined organic phases were washed with brine, dried over Na₂SO₄ andthe solvent was removed in vacuo.

The residue was dissolved in 50 ml acetonitrile and 60 mmol silvernitrite were added into a flask protected from light. This suspensionwas stirred for 41 hours at 70° C. After cooling to room temperature thesuspension was filtrated and concentrated in vacuo. The residue wasdissolved in dichloromethane and extracted with Water. The water phasewas washed again with two times with dichloromethane. The combinedorganic phase was washed with water and brine, dried over Na₂SO₄ and thesolvent was removed in vacuo; Yield: 3.05 g (14.5 mmol; 84.5%).

Example 7 Synthesis of 3-Nitrooxy-propyl propionate

9.1 mmol Propionyl chloride were dissolved in 10 ml TMBE and cooled to3° C. 8.25 mmol 3-Nitrooxypropanol and 9.1 mmol triethylamine in 5 mlTMBE were dropped over a period of 5 min at 3 to 6° C. After 2 hours and30 minutes stirring without cooling the reaction mixture were extractedwith 1N HCl, twice with water, washed with brine, dried over Na₂SO₄ andthe solvent was removed in vacuo leaving 1.35 g.

The crude product was purified by flash chromatography on silica gelusing Hexane/Ethyl acetate 4:1; Yield: 1.14 g (6.4 mmol, 78.0%).

Example 8 Synthesis of 3-Nitrooxy-propyl benzoate

16.5 mmol 3-Nitrooxypropanol dissolved in 10 ml TMBE and 18.2 mmolTriethylamine were cooled to 3° C. 18.2 mmol benzoylchloride in 5 mlTMBE were dropped over a period of 7 minutes at 3 to 6° C. After 24hours and 30 minutes stirring without cooling, the reaction mixture wasextracted with sated. NaHCO₃, water, 1N HCl, twice with water, washedwith brine, dried over Na₂SO₄ and the solvent was removed in vacuoleaving 3.3 g.

The crude product was purified by flash chromatography on silica gelusing a gradient of Hexane/Ethyl acetate from 1:0 to 2:1; Yield: 0.66 g(2.9 mmol, 17.7%).

Example 9 Synthesis of 3-Nitrooxy-propyl hexanoate

20 mmol 3-Nitrooxypropanol dissolved in 10 ml Diethylether and 20 mmolTriethylamine were cooled to 0° C. 18.2 mmol hexoylchlorid were droppedover a period of 5 minutes at 0 to 5° C. After 19 hours stirring withoutcooling, the reaction mixture was extracted with 1N HCl, twice withwater, washed with brine, dried over Na₂SO₄ and the solvent was removedin vacuo leaving 3.1 g.

The crude product was purified by flash chromatography on silica gelusing Heptane/Ethyl acetate 4:1; Yield: 2.4 g (10.9 mmol, 60.0%).

Example 10 Synthesis of 3-Nitrooxy-propyl 5-nitrooxy-hexanoate

20 mmol 3-Nitrooxypropanol dissolved in 10 ml Diethylether and 20 mmolTriethylamine were cooled to 0° C. 18.2 mmol 5-nitrooxypentoylchloridwere dropped over a period of 5 min at 0 to 5° C. After stirring overnight without cooling, the reaction mixture was extracted with 1N HCl,twice with water, washed with brine, dried over Na₂SO₄ and the solventwas removed in vacuo.

The crude product was purified by flash chromatography on silica gelusing Heptane/Ethyl acetate 4:1; Yield: 2.4 g (9.1 mmol, 50.0%).

Example 11 Synthesis of Benzylnitrate

10 mmol Benzylbromide dissolved in 80 ml acetonitrile and 25 mmol silvernitrite were added into a flask protected from light. This suspensionwas stirred for 5 hours at 70° C. After cooling to room temperature thesuspension was filtrated and concentrated in vacuo. The residue wasdissolved in dichloromethane and extracted with Water. The water phasewas washed again with two times with dichloromethane. The combinedorganic phase was washed with water and brine, dried over Na₂SO₄ and thesolvent was removed in vacuo; Yield: 1.55 g (10.1 mmol; 100%).

Example 12 Synthesis of 1,3-bis-Nitrooxy-propane

To a solution of 1,3-dibromopropane (2.00 g, 1.0 eq) in 20.0 mL of dryacetonitrile was added Silver Nitrate (3.70 g, 2.2 eq). The reactionmixture was heated at 70° C. for 2 hours in the dark. The resultingmixture was filtered off through celite and the filtrate wasconcentrated. The residue was dissolved into water (50.0 mL), extractedwith dichloromethane (2×50.0 mL), dried over magnesium sulfate andsolvents were evaporated under vacuum to afford 1.44 g of compound as acolorless liquid (Yield=87%).

Example 13 Synthesis of 1,4-bis-Nitrooxy-butane

To a solution of 1,4-dibromobutane (2.00 g, 1.0 eq) in 20.0 mL of dryacetonitrile was added Silver Nitrate (3.50 g, 2.2 eq). The reactionmixture was heated at 70° C. for 2 hours in the dark. The resultingmixture was filtered off through celite and the filtrate wasconcentrated. The residue was dissolved into water (50.0 mL), extractedwith dichloromethane (2×50.0 mL) and dried over magnesium sulphate.Solvents were evaporated under vacuum to afford 1.49 g of compound as acolorless liquid (Yield=89%).

Example 14 Synthesis of 1,5-bis-Nitrooxy-pentane

To a solution of 1,5-dibromopentane (2.00 g, 1.0 eq) in 20.0 mL of dryacetonitrile was added Silver Nitrate (3.30 g, 2.2 eq). The reactionmixture was heated at 70° C. for 2 hours in the dark. The resultingmixture was filtered off through celite and the filtrate wasconcentrated. The residue was dissolved into water (50.0 mL), extractedwith dichloromethane (2×50.0 mL) and dried over magnesium sulphate.Solvents were evaporated under vacuum to afford 1.38 g of compound as acolorless liquid (Yield=82%).

Example 15 Synthesis of 5-Nitrooxy-pentanenitrile

To a solution of 5-bromovaleronitrile (4.00 g, 1.0 eq) in 40.0 mL of dryacetonitrile was added Silver Nitrate (4.60 g, 1.1 eq). The reactionmixture was heated at 70° C. for 2 hours in the dark. The resultingmixture was filtered off through celite and the filtrate wasconcentrated. The residue was dissolved into water (50.0 mL), extractedwith dichloromethane (2×50.0 mL) and dried over magnesium sulphate.Solvents were evaporated under vacuum to afford 3.56 g of compound as acolorless liquid (Yield=99%).

Example 16 In Vivo Effect of 3-Nitrooxypropanol Compared toethyl-3-nitrooxypropionate

Material and Methods

10 sheep were cannulated in the rumen. The trial started one month afterthe surgical operation. There were 3 treatments: control, additive 1 andadditive 2, both at a single dose. Additive 1 isethyl-3-nitrooxypropionate, and additive 2 is 3-nitrooxypropanol of thepresent invention. The experimental design consisted of a 3×3 Latinsquare with 3 sheep per treatment in each period and 3 consecutiveperiods. Each period included 28 days of adaptation to the treatmentplus two consecutive days of methane measurements in chambers andcollection of rumen samples. Over the course of the adaptation phase, amedium term one day methane measurement was done at day 14. In addition,during days 22 and 23 samples of alfalfa hay and oats, placed in nylonbags, were incubated in the rumen of sheep to determine the dry matterruminal degradation. During the two days of methane measurements inchambers (days 29 and 30) rumen contents samples were collected twohours after the morning feeding, sub-sampled and immediately frozenprior DNA extraction and determination of volatile fatty acids andammonia nitrogen concentration. Experimental animals were randomlyallocated in three sub-groups of 3 animals each and were randomlyassigned one of the three treatments (control, additive 1 and additive2). The 3 sub-groups started the adaptation to the diet with a gap oftwo days so they were in the same adaptation day prior methanemeasurement in the chambers. Animals were individually held in cageswith constant access to fresh water. A diet consisting of alfalfa haychopped at 15-20 cm and oats in a 60:40 ratio plus mineral-vitaminsupplement was provided to the animals at approximately 1.1 times theenergy maintenance level in two equal meals at 9′00 and 14′00 hours.Fresh matter intake was monitored daily for each animal throughout thetrial.

The additive was provided twice a day through the ruminal cannula at thesame time as the feed. The corresponding amount to each additive (100 mgper animal and day for both additives) was pipetted into 10 grams ofgrounded oats and wrapped in cellulose paper immediately before it wasplaced in the rumen. Since the active molecule is volatile thepreviously mentioned procedure was carried out in a cold room at 4° C.

Methane Measurement and Samples Collection

A set of four methane chambers was used. On days 14, 29 and 30 animalswere placed in the chambers for methane measurements. Each chambermeasured 1.8 m wide×1.8 m deep×1.5 m tall. Chamber air temperature wasmaintained between 15 and 20° C. Within each chamber, the animals wereindividually restrained in the same cages as during adaptation.Interruptions occurred daily at 09′00 hours, when the chamber floor wascleaned, and the animals were fed. These interruptions had little impacton the daily methane emissions because fluxes were calculated threetimes per day and then averaged to derive the 23-h emission value.

Airflow and concentration of methane was measured for the inflow andoutflow ducts of each chamber. Air velocity was continuously monitoredover the day in the exhaust duct for each chamber. The air stream ineach of the 4 ducts (chambers 1, 2 and 3 and background) wassub-sampled, and methane concentration was measured continuously using agas analyzer ADM MGA3000 (Spurling works, Herts, UK). It took 11 min tosequentially sample the airflow in all inflow and exhausts ducts in thechambers (3 min in chambers 1, 2, 3, 2 min for background). In summary,the flux of methane for each chamber was calculated for each measuringday from the difference of fresh-air inflow and chamber exhaust methaneconcentrations and mean air velocities.

Rumen Samples Analysis

Samples of rumen contents were freeze-dried and thoroughly mixed byphysical disruption using a bead beater (Mini-bead Beater; BioSpecProducts, Bartlesville, Okla., USA) before DNA extraction, which wasperformed from approximately 50 mg of sample using the QIAamp® DNA StoolMini Kit (Qiagen Ltd, West Sussex, UK) following the manufacturer'sinstructions with the modification that a higher temperature (95° C.)was used for lysis incubation. DNA samples were used as templates forquantitative real-time PCR (qPCR) amplification. The abundance of totalbacteria, total protozoa and total methanogenic archaea were quantifiedby Real Time—PCR (qPCR). Different primer sets were used to amplify 16SrRNA gene-targeted total bacteria (Maeda et al., 2003), and 18S rRNAgene-targeted total protozoa (Sylvester et al 2005). Primers designedfor the detection of methanogenic archaea were targeted against themethyl coenzyme-M reductase (mcrA) gene (Denman et al., 2007). Theamplifications mixture contained 11.5 μl 2×RT-PCR supermix BioRad(Bio-Rad Laboratories Inc., Hercules, Calif., USA), 0.4 μl of eachprimer and 0.5 μl of sample in a final volume of 23 μl. Theamplification efficiency was evaluated for each pair of primers with thefollowing program: a 5 min cycle at 95° C., 40 cycles at 95° C. for 15s, 60° C. for 30 s, 72° C. for 55 s and, 75° C. during 6 s forfluorescent emission measures. The melting curve was built by increasingtemperature from 55° C. to 95° C. and readings were taken every 5° C.Amplification of each target group was carried out with the followingprogram: a 5 min cycle at 95° C., 40 cycles at 95° C. for 15 s, 15 s at60° C. and 72° C. for 45 s (including the fluorescence emissionmeasuring) and a melting curve with a set point temperature of 45° C.and end temperature of 95° C. The absolute amount of bacteria, protozoaand methanogenic archaea, expressed as the number of DNA copies, wasdetermined by using the plasmid pCR®4-TOPO (Invitrogen™, Carlsbad,Calif., USA) as standard. The PCR product obtained using the respectiveset of primers was purified and then cloned into pCR® 4-TOPO® plasmid(Invitrogen™, Carlsbad, Calif., USA) to produce recombinant plasmids. Asingle colony, verified for the expected insert using PCR, was grown insolid media with antibiotics and X-gal overnight. Afterwards, ascreening of transformed E. coli colonies was done and some of thepositive ones were randomly selected. After checking the presence of theinserted fragment in the colonies by PCR, massive culture of positivecolonies was done in liquid media overnight. Plasmids belonging to thesecultures were extracted using the Pure Link™ Miniprep kit (Invitrogen™,Carlsbad, Calif., USA) and then sequenced to verify the presence of thefragment inserted. The number of 16S rRNA gene copies present in theplasmid extracts was calculated using the plasmid DNA concentration andthe molecular mass of the vector with the insert. The concentratedplasmid was serially diluted (10-fold) to provide a range of 10⁸ to 10²copies to generate a standard curve.

A relative abundance quantification was used for methanogenic archaeaand protozoa as described by Denman and McSweeny (2006) using the 16sRNAas reference gene. Volatile fatty acids were analysed by gaschromatography and ammonia N concentration by colorimetry following theprotocols established in our laboratory (Martin-Garcia et al., 2004).

Rumen Degradability

Three grams of 2 mm ground feed were placed in 5 cm×10 cm nylon bagswith a pore size of 50 μm (#R510 Ankom in situ bags, Macedon N.Y.). Thetwo ingredients used in the animals' diets were tested: oats and alfalfahay. Bags with oats were incubated in the rumen for 24 hours, whilethose with alfalfa hay for 48 hours. The incubations times were chosenbased on average residing times in the rumen of different feedstuffs. Ondays 22 and 23 two bags per feed and animal and period were. Bags wereplaced in the rumen immediately before the morning feeding. At 24 or 48hours they were taken out of the rumen, washed with cold water andfrozen at −20° C. At the end of every period the frozen bags were washedin a washing machine using a short cold water program including two bagsper feed that had not been incubated in the rumen to account forsolubility. After washing, the bags were placed in the oven at 60° C.for 48 hours. Rumen degradability (%) was calculated as the loss of drymatter over the incubation time.

Experimental Animals Care

All management and experimental procedures to the sheep were carried outby trained personnel in strict accordance with the Spanish guidelines(Act No. 1201/2005 of 10 Oct. 2005) for experimental animal protection.The temperature, humidity and air turn out in chambers were carefullymonitored considering the animal welfare conditions. CO₂ concentrationwas also continuously monitored in order to keep it within the limitsthat ensured a good air quality and renovation rate. Animals didn't showany stressed behaviour while they were allocated in chambers.

Statistical Analysis

Individual methane emissions, VFA profiles, ratio of acetate topropionate, ammonia N concentration, log₁₀ transformations ofconcentration of total bacteria, total protozoa and methanogenic archaeaand the relative abundance were analyzed for effect of including theadditive. The standard error of the mean (SEM) was computed for eachanalysis. Means were further compared using a least significantdifference (LSD) test.

Results

Dry matter intake was not affected (P>0.05) by the treatment and onlyslight reduction in intakes were observed when the animals wereintroduced in the methane chambers on days 14 and 30.

As described for intakes, the body weight (as an average of weightsrecorded prior and after chamber measurements) was not different(P>0.05) among treatments (Table 5). Methane emissions, expressed asliters per kg of fresh matter intake, were significantly (P=0.020)reduced on day 14 when both additives were incorporated in the diet. Thereduction observed against the control was 14% and 23%, respectively,for additives 1 and 2. When methane emissions were recorded two weekslater, on days 29 and 30, there was still a numerically reduction,although it did not reach the statistical significance (P=0.061 and0.183 for days 29 and 30, respectively). If the measurements recordedduring the last two consecutive days are pooled together the effect ofthe addition shows a similar tendency (P=0.092) as the values consideredseparately.

TABLE 5 Effect of the addition of additives 1 and 2 on body weights,intakes and methane emissions by sheep measured on days 14, 29 and 30after commencing the treatment. Addi- P Time Item Control Additive 1tive 2 SEM value day 14 intake, kg/day 0.819 0.849 0.867 CH4 l, day 24.621.9 20.0 CH4 l/kg intake 29.9 25.6 22.5 2.31 0.020 day 29 intake,kg/day 0.856 0.944 0.922 CH4 l, day 22.0 20.9 18.3 CH4 l/kg intake 25.821.7 19.6 2.12 0.061 day 30 intake, kg/day 0.760 0.925 0.747 CH4 l, day22.7 21.8 19.7 CH4 l/kg intake 29.8 23.2 25.6 2.34 0.183 days intake,kg/day 0.780 0.933 0.823 29-30 CH4 l, day 21.8 21.5 19.1 CH4 l/kg intake28.2 22.6 23.1 2.17 0.092 ^(a,b)Values in a row not sharing a commonsuperscript letters significantly differ, P < 0.05. *Average of weighingprior and after chamber measurements. SEM: Standard Error of the Means

TABLE 6 Effect of the addition of additive 1 and 2 on volatile fattyacid profile (mol/100 mol), ammonia N concentration (mg/100 ml) and drymatter degradation (DMD, %) of oats (24 hours) and alfalfa hay (48hours) in the rumen of sheep. Control Additive 1 Additive 2 SEM P valueAcetate 69.2^(c) 67.5^(b) 64.5^(a) 0.742 0.007 Propionate 14.3^(a)16.6^(a) 17.5^(b) 1.030 0.004 Butirate 2.08 2.05 2.11 0.818 0.353iso-butirate 11.2 10.1 12.3 0.201 0.995 Valerate 1.91 1.94 1.82 0.1940.100 iso-valerate 1.47 1.79 1.82 0.281 0.908 Total 57.4 58.2 57.1 5.1930.995 C2/C3 4.91^(b) 4.09^(a) 3.89^(a) 0.262 0.002 N—NH₃ 100.1 97.3104.1 9.157 0.924 DMD alfalfa 78.6 78.3 78.8 1.22 0.725 hay DMD oats74.2 74.0 70.6 2.02 0.167 ^(a,b)Values in a row not sharing a commonsuperscript letters significantly differ, P < 005. SEM: Standard Errorof the Means

The study of the rumen fermentation parameters from rumen samplescollected on days 29 and 30 showed a shift in the fermentation pathways(Table 5) towards a more propionate type profile in the rumen of animalsreceiving both additives in comparison to the control. As a consequence,in both treatments the acetate to propionate ratio was significantly(P=0.002) reduced. The concentration of ammonia N was similar amongtreatments and within the range expected for the diet supplied to theanimals.

The in sacco degradation study on days 22^(nd) and 23^(rd) showed noeffect of the additive treatment on the rumen degradability of bothalfalfa hay and oats.

TABLE 7 Effect of the addition of additives 1 and 2 on the concentration(log copy gene numbers/g fresh matter) of total bacteria (16S rRNA),protozoa (18S rRNA) and methanogenic archaea (mcrΔ gene) in the rumen ofsheep. The relative abundance (ΔCt) in relation to total bacteria isalso shown for protozoa and methanogens. Control Additive 1 Additive 2Error P value Total 7.45 * 10¹⁰ 9.08 * 10¹⁰ 9.74 * 10¹⁰ bacteria log1010.8 10.9 11.0 0.123 0.607 Total 2.84 * 10¹⁰ 1.87 * 10¹⁰ 2.51 * 10¹⁰protozoa log 10 10.4 10.2 10.2 0.212 0.702 ΔCt 1.65 1.58 1.55 0.2670.984 Archaea 3.54 * 10⁸   2.86 * 10⁸   2.86 * 10⁸   log10 8.54 8.458.34 0.133 0.511 ΔCt 0.028 0.022 0.020 0.005 0.602

Total and relative concentration of the analysed microbial groups in therumen showed no difference (P>0.05) among treatments. When the abundanceof both protozoa and methanogenic archaea were expressed relative tototal bacteria the same lack of effect was observed.

Conclusions

The use of both additives resulted in a significant reduction of methaneproduction and, according to the VFA profiles, a shift in the metabolicpathways involved in H₂ transferring was promoted by additives as well.The objective of this trial was to confirm whether the treatment ofanimals for a month showed a persistence of the results observed overtwo weeks treatment. This is essential when assessing the suitability ofthe practical use of a feed additive. In this study both additivesshowed effect over a month treatment in methane emissions that wasfurther confirmed by a shift in the fermentation pattern.

On the other hand, a change in the fermentation pattern might be notonly due to a reduction in methane production but also to a lower fibredegradation which, in turn, would produce less acetate and thereforelowered acetate to propionate ratio. In order to rule out thisoccurring, a rumen degradability assessment was carried out byincubating nylon bags with both oats and alfalfa hay in the rumen of theanimals.

The results showed no such effect on dry matter degradation which isalso supported by the same bacterial and protozoa biomass recorded inanimals receiving the additives compared to those with no treatment.

Example 17 In Vivo Effect of 3-Nitrooxypropanol in Dairy Cows

Material and Methods

Animals: Six rumen fistulated lactating Holstein X Friesian dairy cowsof second or greater parity and weighing from 550 to 800 kg were usedfor the study. Cows were in mid lactation at the start of the study.

Experimental diets: A single total mixed ration (TMR) diet was providedto all cows throughout the study. Cows were fed ad libitum (5% refusals)for the duration of the trial.

Experimental design: Beginning in mid lactation (with milk yields of 30liters or more), the six cows were randomly assigned to one of the threesupplement treatments in a 3×3 Latin Square design (Table 8). Treatmentperiods were 5 weeks in duration.

TABLE 8 Experimental design Cow Pair 1 Pair 2 Pair 3 Square 1 Square 2Cow Cow Period 888 Cow 989 Cow 973 1000 Cow 1030 Cow 1060 1 1 2 3 1 2 32 2 3 1 2 3 1 3 3 1 2 3 1 2 Diets: 1—Control 2—3-Nitrooxypropanol (500mg/day) 3—3-Nitrooxypropanol (2500 mg/day)

Dosing of 3-Nitrooxypropanol or placebo: The doses of 3-Nitrooxypropanolor placebo was administered to the animals via the rumen cannula atfeeding time in the morning and evening.

Period Design: As only two cows can be housed in the indirectcalorimeters at any one time cows were run in pairs staggered by oneweek. At the end of week 4 animals were moved to the indirectcalorimeters and held in individual tie stalls where four complete 24 hrmeasurements of respiratory exchange (methane and carbon dioxideproduction and oxygen consumption) were obtained (Cammell et al., 2000).

Results

Feed Intake: There was no significant effect of the product(3-Nitrooxypropanol) on daily dry matter intake (DMI) (see table 9).

Methane Production: Methane production (liters/d) and methane yield(liters/kg DMI) were significantly reduced by the 3-Nitrooxypropanol.Methane production was 93 and 90% of control values when the 500 and2500 mg/d doses were given, respectively (see table 9). As regardsmethane yield, the corresponding values were 96 and 93% of controlmethane yield, respectively, for the low and high doses.

TABLE 9 Effects of DSM product fed at two doses. Daily dose, mg/d 0 5002500 SEM DMI, kg/d 18.9 18.8 18.5 0.7 CH₄, L/d 594 555 536 15.3 CH₄, g/d425 398 384 11.0 CH₄, L/kg DMI 31.3 29.9 29.2 1.2

Large variations were observed between animals some showing moreresponse that some others. These results show the potential of thecompounds of the present invention in reducing methane production indairy cows, and shed light on further improving the feeding regimen.

The invention claimed is:
 1. A feed composition or feed additive for aruminant comprising an amount between about 1 mg to about 10 g/kg offeed of 3-nitrooxy propanol sufficient to reduce production of methaneemanating from digestive activities of a ruminant by at least 10%calculated in liters per kilogram of dry matter intake when measured ina metabolic chamber.
 2. A feed composition or feed additive for reducingthe production of methane emanating from the digestive activities ofruminants, wherein the feed composition comprises an amount betweenabout 1 mg/kg of feed to about 10 g/kg of feed sufficient to reduceproduction of methane emanating from the digestive activities of aruminant of at least one active organic molecule or a salt thereofselected from the group consisting of 3-nitrooxypropanol,rac-4-phenylbutane-1,2-diyldinitrate,2-(hydroxymethyl)-2-(nitrooxymethyl)-1,3-propanediol,N-ethyl-3-nitrooxy-propionic sulfonyl amide, 5-nitrooxy-pentanenitrile,5-nitrooxy-pentane, 3-nitrooxy-propyl propionate,1,3-bis-nitrooxypropane, 1,4-bis-nitrooxybutane,1,5-bis-nitrooxypentane, 3-nitrooxy-propyl benzoate, 3-nitrooxy-propylhexanoate, 3-nitrooxy-propyl 5-nitrooxy-hexanoate, benzylnitrate,isosorbid-dinitrate, N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide, andbis-(2-nitrooxyethyl) ether.
 3. The feed composition or feed additiveaccording to claim 2 which is a mineral premix, a vitamin premix, or apremix including vitamins and minerals or a bolus.
 4. The feedcomposition or feed additive according to claim 2, wherein the at leastone active organic molecule is 3-nitrooxy propanol.
 5. The feedcomposition or feed additive according to claim 2, wherein the at leastone active organic molecule is a mixture of 3-nitrooxy propanol and1,3-bis-nitrooxypropane.